Effect of Bushen Jianpi Formulation on ER/c-Myc/TERT Signaling Pathway in Premature Ovarian Failure Mice
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摘要: 目的 观察补肾健脾方对卵巢早衰(POF)小鼠ER/c-Myc/TERT信号通路调控的影响。方法 将50只B6AF1小鼠随机分为正常组、模型组、阳性药组、补肾健脾方高剂量组、补肾健脾方低剂量组。采用小鼠透明带3(ZP3)诱导建立POF模型, 各组分别给予药物或生理盐水, 期间定时观察小鼠动情周期的变化。干预4周后, 取小鼠血清和卵巢组织。放射免疫分析法检测小鼠血清雌二醇(E2)、促卵泡生成激素(FSH)的水平; HE染色法观察卵巢组织病理形态; 免疫荧光法检测卵巢透明带的变化; Western blot法检测雌激素受体(ER)、c-Myc和端粒酶逆转录酶(TERT)的表达。结果 与模型组相比, 补肾健脾方高、低剂量组均可显著升高小鼠血清E2水平(P < 0.05), 降低FSH水平(P < 0.05, P < 0.01), 提高生长卵泡和成熟卵泡的数量, 降低卵巢透明带的荧光表达, 显著增加ER、c-Myc、TERT的表达(P < 0.05, P < 0.01)。结论 补肾健脾方通过提高E2水平, 促进ER的激活, 刺激c-Myc的表达, 进而调节TERT的表达, 从而促进卵巢颗粒细胞的增殖发育以及卵母细胞的成熟, 是其发挥治疗POF作用的途径。Abstract:
OBJECTIVE To observe the effect of Bushen Jianpi formulation on ER/c-Myc/TERT signaling pathway in mice with premature ovarian failure. METHODS Fifty B6AF1 mice were randomly divided into normal group, model group, positive group, Bushen Jianpi formulation high-dose group and Bushen Jianpi formulation low-dose group. ZP3 was used to induce the establishment of premature ovarian failure model. Each group was given drug or normal saline, respectively, and the changes of oestrous cycle were observed regularly during the period. After 4 weeks of intervention, serum and ovarian tissue were collected. The levels of serum E2 and FSH were detected by radioimmunoassay. The pathological morphology of ovarian tissue was observed by HE staining. The changes of zona pellucida of ovary were detected by immunofluorescence assay. The expressions of ER, c-Myc and TERT were detected by Western blot. RESULTS Compared with model group, Bushen Jianpi formulation high-dose and low-dose groups could significantly increase serum E2 level, decrease FSH level, significantly increase the number of growing follicles and mature follicles, significantly reduce the fluorescence expression of ovarian zona pellucidum, and significantly increase the expressions of ER, c-Myc and TERT. CONCLUSION Bushen Jianpi formulation can improve the level of estrogen, promote the secretion of ER, stimulate the expression of c-Myc, regulate the expression of TERT, so as to promote the proliferation and development of ovarian granulocytes and the maturation of oocytes, which is the way to play its role in the treatment of premature ovarian failure. -
Key words:
- Bushen Jianpi formulation /
- premature ovarian failure /
- transparent /
- ER /
- c-Myc /
- TERT
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图 1 小鼠动情周期细胞变化图
注: 动情前期大部分是有核上皮细胞, 少量角化细胞, 无白细胞; 动情期基本是角化细胞, 偶有少量上皮细胞; 动情后期是有核上皮细胞、角化细胞、白细胞同时存在; 动情间期大部分是白细胞, 少量上皮细胞和黏液。
表 1 补肾健脾方对小鼠血清E2、FSH水平的影响(x±s, n=10)
组别 剂量/(g·kg-1) E2/(pg·mL-1) FSH/(mU·mL-1) 正常组 — 61.53±16.30 6.94±1.44 模型组 — 44.34±13.23△ 9.41±2.83△△ 阳性药组 0.52×10-3 79.96±13.03** 7.04±1.53* 补肾健脾方
高剂量组17.16 55.58±6.22* 5.30±1.31** 补肾健脾方
低剂量组8.58 56.73±9.53* 6.74±1.29* 注: 与正常组比较, △P < 0.05, △△P < 0.01;与模型组比较, *P < 0.05, **P < 0.01。 
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表 2 各组小鼠卵巢组织形态学比较(x±s，n=5)
组别 剂量/(g·kg-1) 初次级卵泡率/% 成熟卵泡率/% 闭锁卵泡率/% 黄体数 正常组 - 64.81±18.37 7.05±4.44 28.13±15.18 2.20±1.10 模型组 - 32.35±17.91△ 0.71±1.60△ 67.65±17.91△△ 4.60±1.82△ 阳性药组 0.52×10-3 64.34±10.75** 7.80±4.59* 27.85±8.80** 2.00±1.22* 补肾健脾方高剂量组 17.16 57.52±13.89* 4.92±3.07* 37.56±12.69* 1.80±0.45* 补肾健脾方低剂量组 8.58 39.24±16.16 2.87±3.95 57.89±19.68 2.80±0.84 注：与正常组比较，△P < 0.05，△△P < 0.01；与模型组比较，*P < 0.05，**P < 0.01。
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表 3 各组小鼠卵巢ER、c-Myc和TERT相对表达量的比较(x±s, n=6)
组别 剂量/(g·kg-1) ER c-Myc TERT 正常组 — 1.74±0.24 1.70±0.18 1.53±0.33 模型组 — 0.43±0.07△△ 0.66±0.26△△ 0.86±0.08△△ 阳性药组 0.52×10-3 0.87±0.20** 1.22±0.44* 1.09±0.18* 补肾健脾方高剂量组 17.16 0.88±0.25** 1.43±0.48** 1.53±0.43** 补肾健脾方低剂量组 8.58 0.76±0.28* 1.06±0.15** 1.37±0.32** 注: 与正常组比较, △△P < 0.01;与模型组比较, *P < 0.05, **P < 0.01。
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